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Method for visually detecting Hangzhou white chrysanthemum leaf blight chaetomium globosum by using CRISPR/Cas12a system

A technology for Chaetomium globulus and leaf blight, which is applied in the fields of biology and chemistry, can solve the problems of expensive experimental equipment, low sensitivity, long time consumption, etc., and achieves the effects of user-friendliness, low price and high sensitivity

Pending Publication Date: 2022-08-02
CHINA JILIANG UNIV
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Problems solved by technology

The whole process takes 7-10 days and rapid testing is not possible
Or determine the expression level of specific genes by fluorescent quantitative PCR, but this type of detection method requires high technical requirements for instruments and testing personnel, and requires expensive experimental equipment, making it difficult to achieve on-site real-time detection; Immunology uses two specific The real-time fluorescence GHI of the leaf M1N extract and seed soaking solution of naturally infected bacterial blight and streak bacteria were respectively carried out with the sex probe. High, so it is not suitable for mass detection at the grassroots level

Method used

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  • Method for visually detecting Hangzhou white chrysanthemum leaf blight chaetomium globosum by using CRISPR/Cas12a system
  • Method for visually detecting Hangzhou white chrysanthemum leaf blight chaetomium globosum by using CRISPR/Cas12a system
  • Method for visually detecting Hangzhou white chrysanthemum leaf blight chaetomium globosum by using CRISPR/Cas12a system

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Embodiment Construction

[0027] The technical solutions of the present invention will be further illustrated and described below through specific embodiments in conjunction with the accompanying drawings:

[0028] 1. Visual detection of C. globosum by CRISPR / Cas12a system

[0029] The actual samples were pretreated by thermal lysis method. Under sterile conditions, 1 mL of the sample homogenate was taken into a sterile centrifuge tube, centrifuged at 8000 × g for 2 min, and the supernatant was discarded; Centrifuge at ×g for 2 min, discard the supernatant; add 100 μL of sterile deionized water, boil at 100°C for 10 min; centrifuge the thermal lysis solution at 8000 × g for 2 min, and take the supernatant as the substrate for the subsequent amplification reaction (i.e. Target DNA ),like figure 1 for the specific operation steps. The PCR amplification technology is used to amplify the target fragment, and the primers used in the specific gene fragment system are SEQ ID No.1 and SEQ ID No.2.

[0030] ...

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Abstract

The invention provides a method for visually detecting Hangzhou white chrysanthemum chaetomium globosum C.globosum by utilizing a CRISPR / Cas12a (clustered regularly interspaced short palindromic repeats / CRISPR associated 12a) system. The method comprises the following steps: amplifying a specific gene segment of Hangzhou white chrysanthemum chaetomium globosum C.globosum by virtue of a PCR (Polymerase Chain Reaction) technology; under the mediation of crRNA, Cas12a specifically recognizes an amplification product and activates nuclease activity, so that any base sequence ssDNA (Reporter) of which the two ends are modified with FAM and BHQ1 is cut, and an FAM-ssDNA fragment and a BHQ1-ssDNA fragment are formed; the FAM-ssDNA and the BHQ1-ssDNA can show fluorescence under the irradiation of a blue light transmission instrument, so that the detection on the Hangzhou white chrysanthemum chaetomium globosum C.globosum is realized. The method is simple and portable to operate, does not depend on large-scale instruments and equipment, is low in cost, high in sensitivity and strong in specificity, and has a certain application prospect.

Description

technical field [0001] The invention belongs to the fields of biology and chemistry, and relates to a method for visual detection of Chaetomium globosum (C. globosum, Cg) by utilizing CRISPR / Cas12a system. Background technique [0002] Clustered regularly interspaced short palindromic repeats (CRISPR) are an immune mechanism against invasion in many bacteria and archaea. CRISPR and CRISPR associated proteins (Cas) are called CRISPR / Cas system. Cas12a is a class 2 V-type Cas protein. It has no cleavage activity when it is not activated. When it binds to a specific crRNA, the conformation of Cas12a will change, and it will combine with crRNA to form a Cas12a-crRNA binary complex. The complex can specifically recognize the target DNA and activate its endonuclease activity. When Cas12a sequentially cuts DNA double-strands in cis, the active site will continue to be exposed, exhibiting trans-cutting activity, and non-specific cleavage of surrounding ssDNA. According to the cha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/04C12R1/645
CPCC12Q1/6895C12Q1/686C12R2001/645C12Q2521/327C12Q2563/107Y02A50/30
Inventor 宋阳申屠旭萍刘妍俞晓平
Owner CHINA JILIANG UNIV
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